The present invention relates to a novel N-acetylglucosaminyl-transferase (GlCNAc transferase) which recognizes a specific sugar chain structure in a saccharide and introduces thereinto a GlcNAc xcex21xe2x86x924 branching structure.
1. Glycoproteins
Most of proteins occurring in nature are not simple proteins composed of amino acids alone, but xe2x80x9cmaturexe2x80x9d proteins having sugar chains and other substances such as phosphates and lipids attached thereto. Therefore, the development of simple protein-type products produced by Escherichia coli as a host has involved various problems because such products lack the maturing process of proteins. Since all of secretion-type physiologically active proteins (e.g. cytokines) are glycoproteins with a few exceptions, the function and the role of sugar chains have attracted attention as the most important point in the development of biological pharmaceuticals.
Sugar chains in glycoproteins are classified roughly into Asn-linked type, mucin-type, O-linked GlcNAc type, GPI anchor type and proteoglycan type [Makoto Takeuchi, xe2x80x9cGlycobiology Series 5: Glycotechnologyxe2x80x9d, Kihata, Hakomori and Nagai (eds.), Kodansha Scientific Co., (1994), 191-208]. Each of these types of sugar chains has its own biosynthesis pathway and a discrete physiological function. Asn-linked sugar chains are distributed widely in molds, yeasts, insects, plants and animals. The basic biosynthesis pathway for Asn-linked sugar chains is conserved beyond species (FIG. 1). A sugar chain(s) characteristic of a specific species is(are) formed on the outer side (called the xe2x80x9cnon-reducing terminal sidexe2x80x9d) of the core sugar chain moiety which is common in the biosynthesis of Asn-linked sugar chains. A mannan-type sugar chain in whicha xcex1 1,3- and xcex1 1,2-branching mannose residues attach to a main chain extending via xcex1 1,6 linkages is a sugar chain structure characteristic of fungi such as yeasts (see Panel a in FIG. 2) [Hiroshi Nakajima, Sugar Chain Technology, Industry Survey Association (1992), 384-397]. On the other hand, in insects, plants and animals., extension of mannose residues is not observed; instead, a high mannose type sugar chain is formed which is a sugar chain transferred from a dolichol intermediate and only trimmed (see Panel c in FIG. 2). A unique structure having characteristic xylose or the like (see Panel b in FIG. 2) is also observed in insects, plants and mollusks. In animals, characteristic sugar chain structures such as complex type sugar chain (Panel e in FIG. 2) and hybrid type sugar chain (Panel d in FIG. 2) are observed; in the former, GlcNAc branching structures are formed in a once trimmed sugar chain, and addition of other kinds of monosaccharides such as galactose and sialic acid forms complicated structures; in the latter, both a complex type sugar chain and a high mannose type sugar chain are present [Kiyoshi Furukawa, Sugar Chain Technology, Industry Survey Association (1992), 64-75].
Such sugar chains as described above are conferred on most of cell surface proteins and secretion proteins, and are thought to play important roles which determine the natures and properties of cells and proteins. Among all, the portion of a sugar chain structure which forms a branch elongating like antennas from the common core sugar chain is called a sugar chain branching structure. This structure is believed to have a function to give an organism recognition ligand (i.e., the end portion of the sugar chain) a high degree of freedom to thereby provide chances for multipoint recognition and another function to maximize the protection ability for the protein moiety by greatly increasing the space-occupying volume (Takeuchi et al., supra). Therefore, by controlling the branching structure of sugar chains, it is possible to modify the physiological functions, sush as itn vivo stability, in vivo kinetics and organ-targeting properties of glycoproteins in various ways. In view of this, technology to control branching structures of sugar chains is expected as biotechnology of the next generation for the development of glycoprotein-type pharmaceuticals which are xe2x80x9ctender to humansxe2x80x9d.
2. Physiological Significance of Glycoprotein Sugar Chains
Sugar chains of secretion type glycoproteins exhibit excellent functions in biosynthesis, intracellular sorting, masking of antigenicity, in vivo stability and organ-targeting properties of glycoproteins. Sugar chains of cell surface proteins are known to change in response to changes in cells (such as differentiation, change to a morbid state, canceration). In particular, it has been reported that there is a close relation between the metastasis of cancer and the branching structure of sugar chains.
(1) Masking of Antigenicity
It is considered that sugar chains have a high degree of freedom in terms of steric structure and thus are moving freely like propellers. Therefore, protein molecules such as proteases and antibodies against proteins not having affinity to sugar chains are shook off by the sugar chains and thus cannot gain access to the protein moiety. As a result, even if there is antigenicity in the peptide moiety near the sugar chain binding site, antibody molecules cannot have access to the peptide moiety. Thus, an antigen-antibody reaction is extremely difficult to occur. Further, when a glycoprotein has been captured by a macrophage and the degradation products are presented as antigen, receptors are difficult of access to the peptides around the sugar chain binding site. Thus, antigenic stimulation is difficult to occur. Actually, it is reported that when sugar chains have been introduced into the central portion of the antigenic peptide of ovalbumin lysozyme, the binding of MHC class II molecules to the antigen is remarkably inhibited [Mouritsen, S., Meldal, M., Christiansen-Brams, I., Elsner, H. and Werdelin, O., Eur. J. Immunol., (1994), 24, 1066-1072]. The effect of such masking of antigenicity becomes greater as the volume occupied by sugar chains is greater. Thus, it is considered that the development of a branching structure contributes to the effect of such masking greatly.
(2) In Vivo Stability
With respect to erythropoietin which is the first glycoprotein-type pharmaceutical ever produced from a transgenic animal cell as a host, the functions of sugar chains thereof have been studied thoroughly. As a result, it has been shown that the sugar chains of erythropoietin work inhibitorily against the binding of erythropoietin with its receptor but make a decisive contribution to the retaining of the active structure and the improvement of in vivo kinetics; as a whole, the sugar chains have been shown to be essential for expression of the pharmacological activity of erythropoietin (Takeuchi, M. and Kobata, A., Glycobiology (1991), 1, 337-346). In particular, a strong correlation between the number of antennae in sugar chains and the pharmacological effect of erythropoietin has been found, and thus the importance of its branching structure (a branching structure formed by GlcNAc residues attaching to the core sugar chain) which never attracted attention has been made clear for the first time [Takeuchi, M., Inoue, N., Strickland, T. W., Kobata, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S. and Kobata, A., Proc. Natl. Acad. Sci. USA, (1989), 86, 7819-22]. The major cause of the above phenomenon is explained as follows: erythropoietin without developed branching structure is cleared rather rapidly in kidney and, as a result, the in vivo residence time of such erythropoietin becomes shorter [Misaizu, T., Matsuki, S., Strickland, T. W., Takeuchi, M., Kobata, A. and Takasaki, S., Blood, (1995), 86, 4097-4104].
(3) Organ Targeting Property
Most of biological tissues have lectin-like receptors and use then in cell-cell interactions or to uptake glycoproteins from blood. The asialoprotein-binding lectin in liver is a representative example of a clearance system for aged glycoproteins [Toshihiro Kawasaki, Sugar Chain Technology, Industry Survey Association (1992), 125-136]. In addition, selectin contained in vascular endothelial cells, platelets and leucocytes (Kawasaki, supra) and the lectin receptor present on the surface of macrophages and NK cells (Kawasaki, supra) are well known. Furthermore, not only glycoproteins but also cells are known to gather in a specific tissue using sugar chains as ligands. Cases of the homing of bone marrow cells [Tatsuo Irimura, xe2x80x9cGlycobiology Series 3: Glycobiology in Cell Societyxe2x80x9d, Katsutaka Nagai, Senichiro Hakomori and Akira Kobata (eds.), Kodansha Scientific Co., (1993), 127-175] and the recruiting of neutrophiles to inflammatory sites (Irimura, supra) are examined in detail. Putting all these things together, it can be well assumed that glycoproteins and cells have, via their sugar chain structures, a targeting property toward specific organs or tissues presenting a lectin receptor in blood circulation, although such a targeting system is not found in all organ. This means that drug delivery by means of sugar chains is possible. In such drug delivery, the affinity of lectin for sugar chains is greatly influenced by the degree of freedom and the number of sugar chain ligands. Therefore, the branching structure of sugar chains will be the most important point in such drug delivery.
(4) Correlation between Cells"" Change into Morbid State and Sugar Chain Branched Structure thereof [Junko Kato, Naoko Suzuki,xe2x80x9d Sugar Chain Technology and Development of Pharmaceuticalxe2x80x9d, Foundation for the Relief and Study of Injury Caused by Pharmaceutical Side Effect (ed.), Yakugyo-Jiho-Sha, (1994), 107-13214]
Once a plant lectin called L-PHA was developed as a probe to detect a multi-branching type sugar chain structure, it has become possible to examine various morbid tissue samples. As a result, a tendency has been found that some types of cancer cells, particularly, cancer cells with a high metastasis ability are stained well with L-PHA. Thus, researchers have become aware of the correlation between the branching structure of sugar chains and the metastasis ability of cancer cells. Human chorionic gonadotropin (hCG) is a glycoprotein hormone vigorously biosynthesized in villus tissues at an early stage of pregnancy. Since a considerable amount of hCG is discharged into urine, hCG is clinically utilized as an indicator of pregnancy. The Asn-linked sugar chains mainly formed by the mono- and biantennary complex type chains are characteristic to hCG. As cancer increases its malignancy from trophoblastoma to invasive mole and from invasive mole to choriocarcinoma, it is reported that 2,4,2 type tri-antennary sugar chains and abnormal biantennary sugar chains (both are formed through the action of GnT-IV on normal biantennary and mono-antennary sugar chains, respectively) appear in the sugar chains of hCG [Katsuko Yamashita, Protein, Nucleic Acid and Enzyme (1992), 37, 1880-1888]. As a cause of this phenomenon, it is suggested that the activity of GnT-IV increases as the malignancy of choriocarcinoma progresses.
xcex3-Glutamyltranspeptidase (xcex3-GTP) is a glycoprotein occurring specifically abundant in liver. Since the serum xcex3-GTP level drastically increases when there is a liver disease, this level is used as clinical indicator of a liver disease. Further, Yamashita et al. [Yamashita, K., Totani, K., Iwaki, Y., Takamisawa, I., Takeishi, N., Higashi, T., Sakamoto, Y. and Kobata, A., J. Biochem., (1989), 105, 728-735] have found that, as a result of canceration of cells, the sugar chain structure of xcex3-GTP changes abnormally in its branching structure similar to those in abnormal hCG; thus, they have reported the correlation between canceration and the activation of GnT-IV. The Asn-linked sugar chains of xcex3-GTP derived from healthy human liver cells are composed mainly of the biantennary complex type sugar chain with small amounts of tri-antennary and tetra-antennary sugar chains mixed therein. In contrast, a remarkable increase in the degree of branching was observed in the Asn-linked sugar chains of xcex3-GTP derived from human hepatoma cells. At the same time, though small in amounts, high mannose type sugar chains and abnormal biantennary sugar chains (both of which were not observed in xcex3-GTP from normal cells) appeared. As a cause of these changes in sugar chain structure, a possibility is suggested that N-acetylglucosaminyltransferase IV (GnT-IV) and V (GnT-V) are activated in relation to canceration of liver cells (Yamashita et al., supra).
It is also reported that the sugar chain branching structure of a glycoprotein in cells is greatly changed by viral infection (Yamashita et al., supra). BHK cells have sugar chain structures with branching up to tetraantennary type. When BHK cells are transformed with polyomavirus, biantennary type sugar chains decrease in the glycoprotein sugar chains produced by the cells, while tetraantennary type sugar chains and the N-acetyllactosamine repeat structures increase; as a whole, a remarkable increase in the number of branches was recognized [Takasaki S., Ikehira, H. and Kobata A., Biochem. Biophys. Res. Commun., (1980), 90, (3), 735-742]. As a cause of the above change, activation of GnT-IV, GnT-V and i-GnT may be considered.
3. Enzymes relating to the Sugar Chain Branching Structures of Glycoproteins
The complex type sugar chain which is a glycoprotein sugar chain structure characteristic of animals has a complicated branching structure in which N-acetylglucosamine (GlcNAc) residues are attaching to the common core structure in various manners (Kiyoshi Furukawa, supra) (FIG. 1). Since this branching structure is closely related to in vivio and in vivo stability, localization, biological activity and pharmacological property of glycoproteins (Makoto Takeuchi, supra), the process of biosynthesis of the branching structure has been investigated in detail. By using inventive substrates H. Schachter et al. have discriminated the various enzyme activities in hen oviduct to thereby predict the presence of GlcNAc branch forming enzymes from GnT-I to GnT-VI (group of GlcNAc glycosyltransferases; FIG. 3) [Glesson, P. A. and Schachter, H., J. Biol. Chem., (1983), 258, 6162-6173]. Thereafter, GnT-I [Kumar, R., Yang, J., Larsen, R. D. and Stanley P., Proc. Natl. Acad. Sci. USA, (1990), 87, 9948-9952; Sarkar, M., Hull, E., Nishikawa, Y., Simpson, R. J., Moritz, R. L., Dunn, R. and Schachter, H., Proc. Natl. Acad. Sci., USA, (1991), 88, 234-238], GnT-II [D""Agostaro, G A., Zingoni, A., Moritz, R L., Simpson, R J., Schachter, H. and Bendiak, B., J. Biol. Chem., (1995), 270, 15211-21], GnT-III [Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K. and Taniguchi, N., J. Biol. Chem., (1992), 267, 18199-18204] and GnT-V [Shorebah, M. G., Hindsgaul, O . and Pierce, M., J. Biol. Chem., (1992), 267, 2920-2927; Gu, J., Nishikawa, A., Turuoka, N., Ono, M., Yamaguchi, N., Kangawa, K. and Taniguchi, N., J. Biochem., (1993), 113, 614-619] were successively purified, and the genes thereof were cloned. However, with these known GlcNAc transferases alone, it is impossible to form the main sugar chain (tetraantennary type; see the formula below) found in xcex1 1 acid glycoprotein known as a representative human blood glycoprotein [Yoshima, K., Tsuji, T., Irimura, T. and Osawa, T, J. Biol. Chem., (1984), 256, 10834-10840] and erythropoietin [Takeuchi, M., Takasaki, S., Shimada, M. and Kobata, A., J. Biol. Chem., (1990), 265, 12127-12130]. Therefore, an N-acetylglucosaminyltransferase having such substrate specificity and reaction specificity that are expected in GnT-IV has been searched for as a missing link. 
In addition to those mentioned above, the following N-acetyl-glucosaminyltransferases have been purified or the genes thereof have been cloned: a transferase acting on mucin-type sugar chains [Bierhuizen, M. F., Maemura, K. and Fukuda, M., J. Biol. Chem., (1994), 269, 4473-4479], a transferase acting on glycolipids, and a transferase forming the sugar chain epitope known as Ixc2x7i antigenic structure [Kawashima, H., Yamamoto, K., Osawa, T. and Irimura, T., J. Biol. Chem., (1993), 268, 27118-27126; Bierhuizen, M. F., Mattei, M. G. and Fukuda, M., Genes Dev., (1993), 7, 468-478]. However, the substrate specificity of these transferases and the mode of binding of the GlcNAc group transferred by these transferases are different from those of GnT-IV. Any of these transferases does not yield products resembling GnT-IV products.
It is an object of the present invention to provide an enzyme having xcex21xe2x86x924 N-acetylglucosaminyltransferase (hereinafter referred to as xe2x80x9cGnT-IVxe2x80x9d) activity; a gene encoding the enzyme; a recombinant DNA comprising the gene; a cell containing the recombinant DNA; a method for producing an enzyme protein having GnT-IV activity comprising culturing the cell in a medium; and a saccharide in which the sugar chains are modified with GnT-IV.
Toward the solution of the above assignments, the present inventors have made intensive and extensive researches. As a result, the inventors have isolated and purified a GnT-IV enzyme protein from bovine small intestine, and characterized the biochemical properties of the protein. Then, the inventors have succeeded in cloning a gene coding for bovine GnT-IVa from a cDNA library and mRNA from the small intestine based on a partial amino acid sequence of the above enzyme protein. Further, based on bovine GnT-IVa gene, the inventors have succeeded in cloning two genes coding for human GnT-IVa and human GnT-IVb, from cDNA libraries and mRNAs from human liver and human lung, respectively. The present invention has been completed by confirming that the products of these genes exhibit GnT-IV activity.
The first invention of the present application relates to a GnT-IV having an activity to produce a saccharide having a partial structure represented by the formula below: 
using UDP-GlcNAc as a sugar donor and a saccharide having a partial structure represented by the formula below as a sugar receptor: 
The second invention relates to a GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 18 or the amino acid sequence shown in SEQ ID NO: 18 which has addition, deletion or substitution of one or more amino acid residues and yet which produces GnT-IV activity; a GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 which has addition, deletion or substitution of one or more amino acid residues and yet which produces GnT-IV activity; and a GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 37 or the amino acid sequence shown in SEQ ID NO: 37 which has addition, deletion or substitution of one or more amino acid residues and yet which produces GnT-IV activity.
The third invention relates to a GnT-IV gene coding for a GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 18 or the amino acid sequence shown in SEQ ID NO: 18 which has addition, deletion or substitution of one or more amino acid residues and yet which produces GnT-IV activity; a GnT-IV gene coding for a GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 which has addition, deletion or substitution of one or more amino acid residues and yet which produces GnT-IV activity; a GnT-IV gene coding for a GnT-IV consisting of the amino acid sequence shown in SEQ ID NO: 37 or the amino acid sequence shown in SEQ ID NO: 37 which has addition, deletion or substitution of one or more amino acid residues and yet which produces GnT-IV activity; a GnT-IV gene consisting of the nucleotide sequence shown in SEQ ID NO: 17; a GnT-IV gene consisting of the nucleotide sequence shown in SEQ ID NO: 23; and a GnT-IV gene consisting of the nucleotide sequence shown in SEQ ID NO: 36.
The fourth invention relates to a recombinant DNA obtainable by inserting any of the above GnT-IV gene into a vector DNA; and a chromosomal fragment comprising a part or all of any one of the above GnT-IV gene.
The fifth invention relates to a host cell carrying the above recombinant DNA; and a host cell into which the above chromosomal fragment is artificially introduced.
The sixth invention relates to a method for producing a GnT-IV comprising culturing the above host cell in a medium and recovering the GnT-IV from the resultant culture; and a method for producing a GnT-IV comprising recovering the GnT-IV enzyme from the secreta, body fluids or homogenete originated from the above host cell.
The seventh invention relates to a method for purifying the GnT-IV from biological samples.
The eighth invention relates to a saccharide of which the sugar chain structure is modified with the GnT-IV.
Hereinbelow, the present invention will be described in detail.
The GnT-IV gene of the invention can be isolated as described below.
Isolation of Bovine GnT-IVa Gene
First, a microsome fraction from bovine small intestine solubilized with a detergent is subjected to a series of purification procedures using anion exchange chromatography, copper chelate chromatography, two-step affinity chromatography using a substrate analogue and gel filtration to thereby obtain a purified sample of GnT-IV enzyme. The resultant purified sample is subjected to SDS-PAGE and then transferred onto a PVDF membrane. The transferred protein, as it is or after restricted hydrolysis, is analyzed with a gas phase amino acid sequencer to obtain a partial amino acid sequence for the GnT-IV enzyme.
Subsequently, an RT-PCR is performed on the RNA extracted from the animal cells (i.e., bovine small intestine) as a template using primers designed based on the partial amino acid sequences determined above. Further, using a fragment obtained by the RT-PCR as a probe, the GnT-IV gene of interest is screened from a cDNA library from the above-mentioned tissue by plaque hybridization. A cDNA fragment contained in the resultant positive plaque is cut out and subcloned into a vector such as pUC19, followed by analysis of the nucleotide sequence thereof. If the full length of the gene coding for the protein of interest is not contained in the fragment, plaque hybridization is performed again using a part of the subcloned cDNA fragment as a probe. Alternatively, terminal portions of the cDNA of interest are obtained by RACE or the like based on the information on the nucleotide sequence obtained above. The thus obtained GnT-IV gene (which is named GnT-IVa aferward) is subjected to analysis of its entire nucleotide sequence. Subsequently, the amino acid sequence is translated from the gene having the above-mentioned nucleotide sequence. This amino acid sequence is as shown in SEQ ID NO: 18.
Isolation of Human GnT-IVa and GnT-IVb Genes
Human GnT-IVa and GnT-IVb genes can be obtained by performing a RT-PCR using RNA extracted from a human tissue (liver or lung) and based on the information on the nucleotide sequence of bovine GnT-IVa gene as obtained above, followed by screening of a cDNA library from the above tissue. The resultant human GnT-IVa and GnT-IVb genes are subjected to analysis of their entire nucleotide sequences. Subsequently, the amino acid sequences are translated by these genes. These amino acid sequences are as shown in SEQ ID NOS: 24 and 37.
In order to obtain a DNA coding for the amino acid sequence shown in SEQ ID NO: 18, 24 or 37 having addition, deletion or substitution of one or more amino acid residues, a number of methods may be used. For example, a method of treating DNA with a mutagen to induce point mutation or a deletion mutation; a method comprising cleaving DNA selectively, removing or adding a selected nucleotide and then ligating DNA; site-specific mutagenesis; and the like may be enumerated.
The GnT-IV protein of the invention can be produced by preparing a recombinant vector into which a DNA coding for the GnT-IV of the invention obtained by the method described above is inserted downstream of a promoter, introducing the vector into a host cell and culturing the resultant cell. The vector DNA used for this purpose may be either plasmid DNA or bacteriophage DNA. For example, pSVL vector (Pharmacia, Sweden) shown in an Example described later may be used. As the host cell into which the resultant recombinant DNA is introduced, any cell that is conventionally used in recombinant DNA techniques may be used, for example, a prokaryotic cell, an animal cell, a yeart, a fungi, an insect cell. Specific examples include Escherichia coli as a prokaryotic cell and CHO cells from chinese hamster or COS cells from monkey as an animal cell.
The transformation of the host cell described above is performed by conventional methods for each host. For example, if the host is E. coli, a vector comprising the recombinant DNA is introduced by the heat shock method or electroporation into competent cells prepared by the calcium method or the like. If the host is yeast, a vector comprising the recombinant DNA is introduced by the heat shock method or electroporation into competent cells prepared by the lithium method or the like. If the host is an animal cell, a vector comprising the recombinant DNA is introduced into the cell at the growth phase or the like by the calcium phosphate method, lipofection or electroporation.
By culturing the thus obtained transformant in a medium, the GnT-IV protein is produced.
In the cultivation of a transformant, any medium may be used as long as the host is viable in it. For example, LB medium or the like may be used if the host is E. coli. If the host is yeast, YPD medium or the like may be used. If the host is an animal cell, Dulbecco""s medium supplemented with an animal serum or the like may be used. The cultivation is performed under conditions conventionally used for the host. For example, if the host is E. coli, cells are cultured at about 30-37 xc2x0 C. for about 3-24 hours with, if necessary, aeration and/or agitation. If the host is yeast, cells are cultured at about 25-37 xc2x0 C. for about 12 hours to 2 weeks with, if necessary, aeration and/or agitation. If the host is an animal cell, cultivation is performed at about 32-37 xc2x0 C. under 5% CO2 and 100% humidity for about 24 hours to 2 weeks with, if necessary, change of the aeration conditions and/or agitation.
After the cultivation, the cultured microorganism or cells are disrupted using a homogenizer, French press, sonication, lysozyme and/or freeze-thawing to thereby elute the GnT-IV protein outside the microorganism or cells. Then, the protein can be obtained from soluble fractions. If the protein of interest is contained in insoluble fractions, the insoluble fractions are collected by centrifugation after disruption of the microorganism or cells. Then, the protein may be solubilized with a buffer containing guanidine hydrochloride or the like for recovery. Alternatively, the cultured microorganism or cells may be disrupted directly with a buffer containing a protein denaturing agent such as guanidine hydrochloride to thereby elute the protein of interest outside the microorganism or cells.
Purification of the GnT-IV protein from the above supernatant may be performed by the method described in Example 1. Alternatively, this purification may be performed by appropriately combining conventional separation/purification methods. These conventional separation/purification methods include, but are not limited to, centrifugation, salting out, solvent precipitation, dialysis, ultrafiltration, partition chromatography, gel filtration, capillary electrophoresis, TLC, ion exchange chromatography, metal chelate chromatography, affinity chromatography, reversed phase chromatography and isoelectric focusing.
The biochemical properties of the GnT-IV enzyme protein obtained from bovine small intestine as described above are as follows.
(1) Action
This enzyme protein produces a saccharide having a partial structure represented by the formula below: 
using UDP-GlcNAc as a sugar donor and a saccharide having a partial structure represented by the formula below as a sugar receptor: 
The saccharide as a sugar receptor means an oligosaccharide, polysaccharide, glycoconjugate (glycopeptide, glycoprotein, glycolipid or proteoglycan) or a derivative thereof.
(2) Substrate Specificity
When the sugar receptor is an oligosaccharide (for the structures of oligosaccharides, see FIG. 4), the enzyme protein exhibits reactivities of 0% toward core type oligosaccharides, 54% toward GnT-I product type oligosaccharides and 164% toward GnT-V product type oligosaccharides, wherein the reactivity of the enzyme protein toward GnT-II product type oligosaccharides is regarded as 100%.
The enzyme protein exhibits a reactivity of 46% toward a structure of GnT-II product type oligosaccharides in which fucose is attached via xcex11xe2x86x926 linkage to the GlcNAc at the reducing terminus.
The enzyme protein exhibits a reactivity of 0% toward a structure of GnT-II product type oligosaccharides in which the GlcNAc on the xcex11xe2x86x923 mannose is lacking.
The enzyme protein exhibits a reactivity of 16% toward a structure of GnT-II product type oligosaccharides in which galactose is attached viag xcex21xe2x86x924 linkage to the GlcNAc on the xcex1 1xe2x86x926 mannose, and a reactivity of 0% toward a structure of GnT-II product type oligosaccharides in which galactose is attached via xcex21 4 linkage to the GlcNAc on the xcex11xe2x86x923 mannose.
The enzyme protein exhibits a reactivity of 0% toward a structure of GnT-II product type oligosaccharides in which GlcNAc is attached via xcex2 1xe2x86x924 linkage to the xcex21xe2x86x924 mannose.
(3) Molecular Weight
About 66 K as determined by SDS-PAGE (under non-reducing conditions). About 60 K after treatment with peptide N-glycosidase F. Since a shift of band is observed when peptide N-glycanase is used, the enzyme protein is thought to be a glycoprotein.
The apparent molecular weight as determined by filtration with a gel containing Triton X-100 is 77 K. Thus, it is thought that GnT-IV does not have a subunit structure and functions as a monomer.
The protein moiety of this enzyme deduced from the nucleotide sequence thereof consists of 535 amino acid residues and has a molecular weight of 61614.
(4) Optimum pH
The optimum pH for reaction is about 5.5. More than 50% of the maximum activity is observed in the range from pH 6.5 to 8.0.
(5) Inhibition, Activation and Stabilization
(i) Inhibition
The activity of this enzyme is inhibited by addition of 20 mM EDTA.
This enzyme is inhibited by UDP derivatives. The intensity of inhibition is in the following order: UDP greater than  greater than UDP-Glc greater than UDP-GalNAc greater than  greater than 2xe2x80x2-deoxy UDP greater than UDP-hexanolamine greater than  greater than UDP-Gal greater than UTP greater than UDP-glucuronic acid greater than UMP.
Uridine, TDP and CDP do not have inhibitory effect.
(ii) Activation
A divalent cation is essential for expression of the activity. Among divalent cations, Mn2+ shows the greatest effect. At a concentration of 7.5 mM, the respective effects of Co2+and Mg2+ are about 70% of that of Mn2+, and the effect of Ca2+ is about 10% of that of Mn2+. The effect of Mn2+ is greatest in the range from 5 to 20 mM.
(iii) Stabilization
Stabilizing effect is recognized in BSA and glycerol.
(6) Kinetic Parameters
When the saccharide as a receptor is an oligosaccharide (for the structures of oligosaccharide, see FIG. 4):
(i) under assay conditions in which the enzyme is reacted in 50 xcexcl of 125 mM MOPS buffer (pH 7.3) containing 0.8 mM receptor substrate, 20 mM UDP-GlcNAc, 7.5 mM MnCl2, 200 mM GlcNAc, 0.5% (w/v) Triton X-100, 10% glycerol and 1% BSA at 37xc2x0 C. for 4 hours:
Km and Vmax values toward GnT-II product type oligosaccharide are 0.73 mM and 3.23 xcexcM/min, respectively.
Km and Vmax values toward GnT-V product type oligosaccharide are 0.13 mM and 1.75xcexcM/min, respectively.
When GnT-II product type oligosaccharide is the receptor substrate, Km value toward UDP-GlcNAc is 0.22 mM.
(ii) under assay conditions in which the enzyme is reacted in 125 mM MOPS buffer (pH 7.3) containing 120 mM UDP-GlcNAc, 7.5 nM MnCl2, 0.5% (w/v) Triton X-100, 10% glycerol and 1% BSA at 37xc2x0 C. for 4 hours:
Km and Vmax values toward GnT-II product type oligosaccharide are 0.59 mM and 0.74 mM/min/mg, respectively.
Km and Vmax values toward GnT-V product type oligosaccharide are 0.14 mM and 0.47 mM/min/mg, respectively.
(7) GnT-IV Family
The homology between bovine GnT-IVa and human GnT-IVa is 91% at the nucleic acid level and 96% at the amino acid level.
All of the partial amino acid structures contained in the purified GnT-IV from bovine small intestine are encoded in the bovine GnT-IVa gene.
Human GnT-IVb and human GnT-IVa have 63% homology at the nucleic acid level and 62% homology at the amino acid level. However, they are entirely different in the C-terminal and N-terminal regions.
From the biochemical properties described above, the GnT-IV of the invention has been recognized as a novel enzyme in the point that this enzyme is able to perform the following reaction which conventional enzymes cannot perform: 